CN106749639A - A kind of new H5 subtype avian influenzas Yolk antibody replaces the method for serum antibody monitoring - Google Patents
A kind of new H5 subtype avian influenzas Yolk antibody replaces the method for serum antibody monitoring Download PDFInfo
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Abstract
The invention belongs to Yolk antibody monitoring technical field, and in particular to a kind of new H5 subtype avian influenzas Yolk antibody replaces the method for serum antibody monitoring.The present invention provides one kind and replaces blood serum sample using egg yolk samples, and extracting yolk liquid and chloroform recovery yolk liquid using phosphate buffer carries out the method that antibody detection is monitored instead of serum antibody.Characterized in that, need not be taken a blood sample during antibody detection, it is only necessary to collect birds, beasts and eggs, will not to poultry produce stress, do not interfere with poultry health and production performance；Replace serum antibody titer after revision with two kinds of mixed liquor H5 subtype avian influenza antibody titers of yolk treatment fluid during antibody detection, it is mainly used in egg fowl and plants the H5 subtype avian influenza antibody detections of fowl, it can also be used to assesses chick epidemic disease maternal antibody level to determine First immune time.
Description
Technical field
The invention belongs to Yolk antibody monitoring technical field, and in particular to a kind of new H5 subtype avian influenza Yolk antibody generations
For the method for serum antibody monitoring.
Background technology
H5 subtype avian influenzas belong to highly pathogenic bird flu, are one of fowl infections of most serious, can cause aviculture
The heavy economic losses of (including chicken, duck, dove, quail etc.), immunity inoculation is the topmost prevention and control measure of China, is immunized what is produced
Antibody titer height directly decides immune quality and prevention and control success or failure.Because immunity inoculation is easily affected by various factors,
Such as vaccine factor, animal factor, feeding and management factor, disease factor, and the immune response of animal can't see, can not touch,
So immune quality has very big uncertainty, this brings great difficulty to prevention and control.Antibody detection is the immune quality of inspection
Most directly, most efficient method, the immune programme for children of science, rational immunization time can be determined by antibody detection, selected excellent
The vaccine of matter, accurate immunizing dose, so that it is guaranteed that immune effect.Antibody detection can also point out what H5 subtype avian influenzas occurred
Risk, if antibody titer is less than 4log2, the antibody of chicken group is insufficiently resistant to the invasion and attack of virus, and the risk of morbidity increases, periodically
Carrying out H5 subtype avian influenzas antibody detection can assess the generation of vaccine immunity quality and early warning epidemic disease, be prevention and control H5 hypotypes
The important measures of bird flu.
Antibody detection is the method that corresponding specific antibody is detected with known antigens using the principle of immunological testing, its
Played a significant role at aspects such as immune quality evaluation, disease diagnosis, Disease monitor, epidemic situation early warning, Blight controls, be animal epidemic disease
The technical way of sick prevention and control.Antibody detection is main with serum antibody titer as standard, and serum is detected using serological test
Antibody, conventional method has hemagglutination test (HA), hemagglutination-inhibition test (HI) and EUSA (ELISA) etc., its
Middle HA and HI are the most popular methods of avian influenza antibody monitoring.Serum antibody monitoring has that result is accurate, reliable advantage, but together
When have the shortcomings that it is cumbersome, influence chicken health and production performance.Since it is desired that taking a blood sample and separating serum, operating process is numerous
It is trivial, waste of manpower and time, while stress being had undesirable effect to poultry production of causing of blood sampling, so as to cause serum antibody
Monitoring is subject to certain restrictions in practical application.However, compared with prepared by serum, yolk preparation is easier and safe, only
Need to collect egg, it is not necessary to take a blood sample, so replacing serum to carry out antibody detection with yolk has certain advantage and promotion price
Value, China carries out the report of newcastle epidemic disease antibody monitoring with regard to useful Yolk antibody early in the eighties in 20th century instead of serum antibody.
Yolk antibody (IgY) is birds produced under antigenic stimulus be deposited on yolk in for the anti-of specific antigen
Body, is the material for improving chick resistance that birds are formed during long-term evolution.When poultry is pierced by exotic antigen
After swashing, the B cell differentiation in the bursa of farbricius turns into thick liquid cell, and secreting specificity antibody enters blood circulation；When blood flows through ovary
When, optionally (mainly IgG) is shifted and is deposited to yolk specific antibody, forms Yolk antibody；IgG has in egg cell
There is cumulative effect, so the concentration of Yolk antibody is higher than the AC in serum.Chicken was reported first from KIemperer in 1893
Since there is antibody in egg, people constantly deepen to the understanding of Yolk antibody, and correlative study also more and more attracts attention, wherein using
It is wherein one of content that Yolk antibody carries out antibody detection instead of serum antibody.It is currently used in the side of detection avian influenza egg yolk antibodies
Method has various, such as chloroform extraction, physiological saline extraction method, phosphate buffer extraction method, and wherein chloroform extraction is used
At most.Although the method that chloroform extraction is closest to serum antibody, result is still present asking for Stability and veracity
Topic, this is that Yolk antibody carries out the maximum problem of avian influenza antibody monitoring instead of serum antibody.Although Yolk antibody monitoring has
Easy to operate, safe advantage, but have that result is inaccurate, insecure shortcoming simultaneously.
Yolk antibody refers to the birds specific antibody that yolk is contained within after being immunized；Serum antibody is birds after immune
The specific antibody that serum is contained within；Hemagglutination-inhibition test refers to that some have virus (or antigen) energy of aggegation red blood cell ability
Suppressed by corresponding antibodies, principle is after corresponding antibodies are combined with viral (or antigen), to prevent viral (or antigen) surface hemagglutinin
With erythrocyte binding, it is usually used in orthomyxovirus, the diagnosis of paramyxovirus and antibody detection；Antibody detection is by immunological experiment
Method, i.e., with known antigens detect test sample in whether there is corresponding specific antibody, its Main Function be inspection immunity
With immune effect, for finding that immune effect is bad in antibody detection, can point out to need booster immunization or adjustment immune programme for children,
Be important technological means in animal epidemic Prevention and control, possess important reference value, and the diagnosis of non-diseases with control
Treatment method.
In the antibody detection of birds epidemic disease, Serum Antibody Detection reliable results are it is well known that still serum antibody monitoring
Need blood sampling, have certain technical difficulty aborning, can also produce stress, influence poultry health and production performance；Yolk
Antibody test need not take a blood sample, it is only necessary to provide birds, beasts and eggs, will not produce stress, do not interfere with poultry health and production performance.
The antibody detection of ewcastle disease and avian influenza virus is always with serum antibody titer as standard, and Yolk antibody potency and serum antibody
There is a certain distance in potency, causing Yolk antibody to detect cannot approve aborning.Generate in recent years it is many wish with
The method that Yolk antibody monitors alternative serum antibody detection, but the situation of Yolk antibody has certain gap with serum antibody, it
Between relevance be difficult to ensure that, often lead to monitoring result unreliable.Prior art has a lot for relevance between the two
Analyzed, but result is often limited only to measure the numerical value contrast of potency, it is intended to therefrom sum up simple numerical value
Rule is come, and to be intended to enable that the monitoring of Yolk antibody replaces serum antibody, but this is that deficiency is won the confidence.The effect of Yolk antibody
The determination of valency is the most critical point that yolk replaces serum, determines that technology is highly developed compared to the potency of serum antibody, and yolk resists
Body is present in yolk, exists in egg white, heterogeneous liquid object mixing is belonged to again, it is difficult to ensure that yolk in existing technology
In all antibody both participated in antibody detection experiment, be frequently not to introduce less according to the difference for actually taking method, be exactly
Antibody is miscalculated to more, single method is it is difficult to ensure that the credibility of the Yolk antibody potency of gained.
The content of the invention
Applicants experimentally found that, the monitoring of H5 subtype avian influenzas antibody titer is carried out for yolk, process one with chloroform
As potency it is relatively low, and higher with the general potency of water process, applicant thinks that it is based on following mechanism：
Contain moisture about 48.0%, protein about 17.8% and fat about 30.5%, wherein most protein in yolk
Combined with fat and existed in the form of lipoprotein, including Yolk antibody.Just because of Yolk antibody is difficult simple separation, so
It is difficult to survey quasi- Yolk antibody potency in practical measurement.
Chloroform method for commonly using is extracted and surveys potency, and chloroform can dissolve the fat in yolk, and the yolk processed with it is passed through
It is divided into 3 layers after centrifugation, orlop is faint yellow, is chloroform layer；Intermediate layer is the fat deposit of solidification；The superiors' clear, to carry
Pure Yolk antibody solution.In processing procedure, meeting lost part Yolk antibody is lost in dissolving in fat deposit or chloroform layer
Fat in, so HI antibody titer can be caused relatively low.
And another treatment route, water-soluble treatment fluid, such as physiological saline, liquor sodii citratis, phosphate buffer,
Treatment yolk primarily serves simple diluting effect, then, in practical measurement, except antibody and antigen occur specific binding with
Outward, other compositions of yolk also there occurs that nonSing ularity is combined with antigen, so HI antibody titer measured value can be caused higher.
And in the prior art, the monitoring for serum antibody titer is typically relatively more accurate, because the separation of Serum Antibody
Technology maturation, accurately, reproducibility is strong, although monitoring sample with Yolk antibody collects simple, fowl advantage only is not influenceed, but
Have the shortcomings that measurement result is inaccurate, do not overcome this to have the disadvantage to be difficult alternative serum antibody detection.
The present invention is it is therefore contemplated that by the Yolk extraet of the Yolk extraet of chloroform treatment and phosphate buffer treatment
Merge, thus to both Technique deviations higher and relatively low respectively of punching, so as to obtain reliable Yolk antibody potency, antibody
Potency again can be closer to the antibody titer of serum through revision.Antibody detection plays an important roll in prevention and control, aborning will
More and more applied, the present invention is the blood and egg of correspondence collection commodity egg, detected that serum, phosphate delay respectively
Fliud flushing extracts the H5 subtype avian influenza HI antibody titers of yolk liquid and chloroform recovery yolk liquid, is carried in phosphate buffer
On the basis of yolk liquid and chloroform recovery yolk liquid potency numerical value are taken relative to serum antibody offset direction objective analysis, one is set up
The method that kind of Yolk antibody carries out antibody detection instead of serum antibody, solves that Yolk antibody result is inaccurate, insecure asks
Topic.The method of the present invention is easy to operate, safety, as a result accurately, reliably, serum can be replaced to carry out egg fowl, plant fowl with yolk
H5 subtype avian influenza antibody avian influenza virus antibody is monitored, it is to avoid the trouble of blood sampling and influence, it is easy to promotion and application.
The method of the present invention is the analysis based on such objective circumstances, that is, cause two kinds of common method treatment yolk to supervise
The method for surveying potency gives complementation, refers here to chloroform and uses phosphate buffer.
The present invention provides one kind and replaces serum sample using yolk sample, and yolk liquid and chlorine are extracted using phosphate buffer
The imitative yolk liquid that extracts carries out method of the H5 subtype avian influenzas antibody detection instead of serum antibody monitoring.Characterized in that, antibody is supervised
Need not be taken a blood sample during survey, it is only necessary to collect birds, beasts and eggs, will not to poultry produce stress, do not interfere with poultry health and production
Performance；Replace serum antibody through further revision value with two kinds of mixed liquor antibody titers of Yolk extraet during antibody detection
Potency, is mainly used in egg fowl and plants the H5 subtype avian influenza antibody detections of fowl, it can also be used to assess chick fowl H5 subtype avian influenzas
Antibody level is determining First immune time.
The beneficial effects of the invention are as follows：Using birds, beasts and eggs as detection sample, yolk liquid and chloroform are extracted with phosphate buffer
Yolk liquid is extracted, two kinds of extract solutions carry out mixed in equal amounts, the H5 subtype avian influenzas antibody titer for carrying out mixed liquor is determined, through appropriate
Replace serum antibody titer after revision.Revisory coefficient is 0~1, is by factor shadows such as Vaccines classes, immunization period, immune programme for children
Ring.With it, antibody detection operation is easier, safer, and it is as a result more accurate, more reliable, greatly improve H5 hypotypes fowl stream
Sense antibody detection applicability aborning.
The method of H5 subtype avian influenza antibody in a kind of claimed new detection yolk, it is characterised in that：
1) yolk feedstock capture step
Egg collection is 20 pieces of random acquisition egg or more in poultry-farm, it is ensured that the egg of collection is from whole chicken group
Decentralization；Birds, beasts and eggs tweezers are gently knocked open the duck eye of diameter about 1cm on shell, shell membrane is torn and is poured out egg white completely, then
Open eggshell and yolk completely poured into culture dish, and then choose out yolk bag, surface egg white is directly avoided with syringe and extracts yolk,
The yolk that will be drawn into is injected into small beaker, and glass rod is stirred evenly makes all extraction yolk fully mix, standby.
2) Yolk antibody is extracted
A, phosphate buffer extract antibody from yolk
Draw in 2ml extraction yolk addition centrifuge tubes, add 2ml phosphate buffers, fully vibration is mixed, and room temperature is put
1h is put, the Yolk antibody extract solution A of muddiness is obtained, it is standby.
B, chloroform recovery antibody from yolk
Draw in 2ml extraction yolk addition centrifuge tubes, add 2ml phosphate buffers, 4ml chloroforms are added after mixing, fill
Divide vibration to mix, room temperature places 1h, period stirs and evenly mixs 3 times, then 10min, appearance layering, upper strata are centrifuged by 3000r/min
It is Yolk antibody extract solution B, separates standby.
3) two kinds of mixing of extracting mode yolk liquid
Yolk antibody extract solution A obtained by step 2 is mixed into obtain mixed liquor, as monitoring ovum with B by equal weight proportions
Yellow antibody sample.
4) hemagglutination test
On Microhemagglutination plate, from the 1st row row of Kong Zhi 12 hole, 25 μ l PBS are added per hole, draw certain multiple dilution
Antigen 25 μ l be added on the 1st row hole, removed after being sufficiently mixed 25 μ l to the 2nd row hole, the like do equivalent doubling dilution to the 11st
Row hole, the 11st row hole discards 25 μ L；The 12nd row hole is set up for red blood cell is compareed；Per the μ l PBS of Kong Zaijia 25；Added per hole
The μ l of 0.75% chicken erythrocyte suspension 25；Put immediately and mix 1min on microoscillator, or hand-held blood-coagulation-board mixes 1min around circle；
30min is acted at room temperature, or acts on 60min at 4 DEG C；Observe for several times, until the red blood cell of control is all precipitated as one
Round dot, blood-coagulation-board is inclined, and observation red blood cell whether there is teardrop sample sagging, according to blood clotting spectral discrimination result；Hemagglutinative titer is higher than
Can continue to increase the hole count of dilution when 211, with the hemagglutinative titer that the antigen greatest dilution for complete aggegation occur is the antigen, this
When hemagglutinative titer be 2n, n>11, the HAU of antigen is calculated further according to initial dilution；And according to the blood clotting of the antigen for determining
Potency, calculates 4 extension rates of HAU antigen, prepares 4 HAU antigens.
5) hemagglutination-inhibition test confirms with potency
On Microhemagglutination plate, from the 1st row row of Kong Zhi 12 hole, 25 μ l PBS are added per hole.
By the mixed liquor of step 3, take the μ l of volume 25 and arrange hole in the 1st, after mixing from left to right order doubling dilution to the 11st
Row hole, finally discards 25 μ l；The 1st row row of Kong Zhi 12 hole adds 25 μ l containing 4 antigens of HAU respectively；Miniature shaking is put immediately
Swing and mix 1min on device, or hand-held blood-coagulation-board mixes 1min around circle；30min is acted at room temperature, or is acted at 4 DEG C
60min；Add the μ l of 0.75% chicken erythrocyte suspension 25 per hole；The rearmounted effect 40min at room temperature of mixing acts on 60min at 4 DEG C,
The red blood cell for now compareing has been precipitated as a round dot；Blood clotting suppresses to continue to increase the hole count of dilution when potency is higher than 211, with
The maximum dilution multiple for completely inhibiting RCA is that the blood clotting of the mixed liquor suppresses potency, determines antibody titer plus revision
The blood clotting that coefficient is Yolk antibody alternative serum antibody suppresses potency.
6) main agents and preparation
PBS：
Prepare 20 times of PB：5.157g Na2HPO4.12H2O and 0.874g NaH2PO4.2H2O are weighed, plus distilled water is extremely
100ml；Prepare 1 times of PBS：Measure 20 times of PB of 50ml, add 8.5g NaCl, plus distilled water is to 1000ml；Adjusted with NaOH or HCl
PH value is to 7.2；121 DEG C of high pressure steam sterilization 15min.
0.75% chicken erythrocyte suspension：
At least 3 SPF cocks of collection or the blood and isometric Alsever's Solution of the healthy cock without H5 subtype avian influenza antibody
Mixing, is washed 3 times with foregoing PBS liquid, and 5-10min is centrifuged with 1000r/min every time, is made into foregoing PBS after washing
0.75% red cell suspension, 4 DEG C save backup, and described 0.75% is volume ratio.
Foregoing preparation method, it is characterised in that：
Wherein described antigen is H5 subtype avian influenza antigens；
In foregoing step 3 by equal weight proportions, be Yolk antibody extract solution A：50%, Yolk antibody extract solution B：
50%；
Foregoing preparation method, chicken erythrocyte suspension therein can now with i.e. use, it is also possible to utilizes alserver's solution
Prepare, the red cell preservation Alsever's Solution compound method：Claim glucose 2.05g, sodium citrate 0.8g, citric acid 0.055g, chlorination
Sodium 0.42g, plus distilled water is to 100ml, and pH value is adjusted after dissolving to 6.1,115 DEG C of high pressure steam sterilization 20min, 4 DEG C save backup.
Brief description of the drawings
Fig. 1 is the preparation side of the Yolk antibody mixed liquor of the replacement H5 subtype avian influenzas serum antibody monitoring that the present invention is provided
The flow chart of method.
Fig. 2 is the computational methods of the Yolk antibody sample of the replacement H5 subtype avian influenzas serum antibody monitoring that the present invention is provided
Flow chart.
Specific embodiment
Following examples will be helpful to one of ordinary skill in the art and further understand the present invention, but not in any form
The limitation present invention.
Embodiment
1) yolk feedstock capture step
Egg collection is 24 pieces of the random acquisition egg in laying hen, it is ensured that dispersion of the egg of collection from whole chicken group
Degree；Birds, beasts and eggs tweezers are gently knocked open the duck eye of diameter about 1cm on shell, shell membrane is torn and is poured out egg white completely, then open egg
Yolk is completely poured into culture dish by shell, and then chooses out yolk bag, surface egg white is directly avoided with syringe and extracts yolk, will be extracted
To yolk be injected into small beaker, glass rod stir evenly make all extract yolk fully mix, it is standby.
2) Yolk antibody is extracted
A, phosphate buffer extract antibody from yolk
It is sufficiently stirred in absorption 2ml extraction yolk addition centrifuge tubes, adds 2ml phosphate buffers, fully vibration is mixed
Even, room temperature places 1h, obtains the Yolk antibody extract solution A of muddiness, standby.
B, chloroform recovery antibody from yolk
Draw in 2ml extraction yolk addition centrifuge tubes, add 2ml phosphate buffers, 4ml chloroforms are added after mixing, fill
Divide vibration to mix, room temperature places 1h, period stirs and evenly mixs 3 times, then 10min, appearance layering, upper strata are centrifuged by 3000r/min
It is Yolk antibody extract solution B, separates standby.
3) two kinds of mixing of extracting mode yolk liquid
Yolk antibody extract solution A obtained by step 2 is mixed into obtain mixed liquor, as monitoring ovum with B by equal weight proportions
Yellow antibody sample.
4) hemagglutination test
On Microhemagglutination plate, from the 1st row row of Kong Zhi 12 hole, 25 μ l PBS are added per hole, draw certain multiple dilution
The μ l of avian influenza antigen 25 be added on the 1st row hole, removed after being sufficiently mixed 25 μ l to the 2nd row hole, the like do equivalent doubling dilution
To the 11st row hole, the 11st row hole discards 25 μ L；The 12nd row hole is set up for red blood cell is compareed；Per the μ l PBS of Kong Zaijia 25；It is each per hole
Add the μ l of 0.75% chicken erythrocyte suspension 25；Put immediately and mix 1min on microoscillator；30min is acted at room temperature；Observation
For several times, until the red blood cell of control is all precipitated as a round dot, blood-coagulation-board is inclined, observation red blood cell whether there is teardrop sample stream
Hang, according to blood clotting spectral discrimination result.According to the hemagglutinative titer of the antigen for determining, 4 dilutions of HAU antigen times are calculated
Number, prepares 4 HAU antigens.H5 subtype avian influenzas (Re-7) hemagglutinative titer is that 210,4 HAU antigens are 28 dilutions.
5) hemagglutination-inhibition test confirms with potency
On Microhemagglutination plate, from the 1st row row of Kong Zhi 12 hole, 25 μ l PBS are added per hole.
By the mixed liquor of step 3, take the μ l of volume 25 and arrange hole in the 1st, after mixing from left to right order doubling dilution to the 11st
Row hole, finally discards 25 μ l；The 1st row row of Kong Zhi 12 hole adds 25 μ l containing 4 avian influenza antigens of HAU respectively；Put immediately
Mix 1min on microoscillator；30min is acted at room temperature；Add the μ l of 0.75% chicken erythrocyte suspension 25 per hole；Mixing is rearmounted
40min is acted at room temperature, and the red blood cell for now compareing has been precipitated as a round dot；To completely inhibit the maximum dilute of RCA
Release the blood clotting that multiple is the mixed liquor and suppress potency, determine antibody titer and be H5 subtype avian influenza ovum plus revisory coefficient 0.8
The blood clotting of yellow antibody surrogate serum antibody suppresses potency.In order to confirm the validity of the inventive method, spy will survey alone chloroform
The result that the yolk numerical value of method and alone phosphate buffer extraction and determination is produced with numerical value of the present invention is simply compared.List
It is as follows：
The 3 kinds of Yolk antibody potency of H5 subtype avian influenzas (Re-7) of table 1 and correspondence serum antibody titer result are (with log2 tables
Show)
Table 1 is the Yolk antibody and correspondence serum antibody result of H5 subtype avian influenzas, and phosphate buffer extracts yolk liquid
Larger with serum antibody titer deviation with chloroform recovery yolk humoral antibody potency, degree of conformity is relatively low, and phosphate buffer, chloroform
The equivalent mixed liquor antibody titer for extracting yolk liquid is smaller with serum antibody titer deviation plus revisory coefficient, and degree of conformity is higher,
It is substantially better than phosphate buffer and extracts yolk humoral antibody potency or chloroform recovery yolk humoral antibody potency, its valence value relatively connects
Nearly serum titer results of measuring, and its operate instrument requirements and tedious steps without serum titer detection, are a kind of
Confidence level is higher, can to a certain extent replace the method for H5 subtype avian influenzas serum antibody monitoring.
6) main agents and preparation
PBS：
Prepare 20 times of PB：5.157g Na2HPO4.12H2O and 0.874g NaH2PO4.2H2O are weighed, plus distilled water is extremely
100ml；Prepare 1 times of PBS：Measure 20 times of PB of 50ml, add 8.5g NaCl, plus distilled water is to 1000ml；Adjusted with NaOH or HCl
PH value is to 7.2；121 DEG C of high pressure steam sterilization 15min.
0.75% chicken erythrocyte suspension：
The blood for gathering 3 healthy cocks without H5 subtype avian influenza antibody mixes with isometric Alsever's Solution, uses foregoing PBS
Liquid is washed 3 times, and 10min is centrifuged with 1000r/min every time, and 0.75% red cell suspension, 4 DEG C are made into foregoing PBS after washing
Save backup, described 0.75% is volume ratio.
In step 3 by equal weight proportions, be Yolk antibody extract solution A：50%, Yolk antibody extract solution B：50%.
Foregoing preparation method, Alsever's Solution formula therein is：
Red cell preservation Alsever's Solution：Claim glucose 2.05g, sodium citrate 0.8g, citric acid 0.055g, sodium chloride 0.42g,
PH value is adjusted plus distilled water is to 100ml, after dissolving to 6.1,115 DEG C of high pressure steam sterilization 20min, 4 DEG C save backup.
Points for attention of the present invention in practical measurement：
(1) potency for testing antigen used must be demarcated accurately, every time certain elder generation's Accurate Determining antigen before monitoring
Potency, it should be to survey to take its average value 2~3 times simultaneously, then according to needed for its accurate potency is prepared antigen liquid (4 units are anti-
It is former).After antigen liquid is prepared, to test oneself once, i.e., in the case of not increase serum, the 1st, the complete aggegation of blood cell in the 2nd hole,
3 bore portion aggegations (blood cell congeals into a red circle rather than uniform aggegation in hole wall), illustrate that the preparation of 4 unit antigens is not asked
Topic, it is possible to use.
(2) red blood cell should pick up from the ripe cock not being immunized, and immature cock red blood cell is more fragile, easy haemolysis,
Can not use.It is more preferable that red blood cell is made into 0.75% after being centrifuged repeatedly washing, in practice it has proved that, 1% red blood cell is all not so good as
0.75% it is good.Red blood cell suspension is preferably now with the current, and the resting period crosses leptocyte and ruptures, and experiment can be influenceed to tie
Really.
(3) revisory coefficient is usually 0~1, is defined as 0.8 in embodiment, revisory coefficient and vaccine, immunization period, immune
Program etc. is relevant, it is necessary to empirically determined, and reliable revisory coefficient can further improve the accuracy of antibody titer.
Presently preferred embodiments of the present invention is the foregoing is only, is not intended to limit the invention, it is all in essence of the invention
Any modification, equivalent and improvement made within god and principle etc., should be included within the scope of the present invention.This
The involved constituent of invention and preparation method can be adjusted accordingly according to implementation method, but can not be protected beyond the invention
Shield scope.
Claims (4)
1. a kind of method that new H5 subtype avian influenzas Yolk antibody replaces serum antibody monitoring, it is characterised in that：
1) yolk feedstock capture step
Egg collection is 20 pieces of random acquisition egg or more in poultry-farm, it is ensured that the egg of collection divides from whole chicken group's
Divergence；Birds, beasts and eggs tweezers are gently knocked open the duck eye of diameter about 1cm on shell, shell membrane is torn and is poured out egg white completely, then open
Yolk is completely poured into culture dish by eggshell, and then chooses out yolk bag, surface egg white is directly avoided with syringe and extracts yolk, will be taken out
The yolk got is injected into small beaker, and glass rod is stirred evenly makes all extraction yolk fully mix, standby；
2) Yolk antibody is extracted
A, phosphate buffer extract antibody from yolk
Draw in 2ml extraction yolk addition centrifuge tubes, add 2ml phosphate buffers, fully vibration is mixed, room temperature placement
1h, obtains the Yolk antibody extract solution A of muddiness, standby；
B, chloroform recovery antibody from yolk
Draw in 2ml extraction yolk addition centrifuge tubes, add 2ml phosphate buffers, 4ml chloroforms are added after mixing, fully shake
Mixing is swung, room temperature places 1h, and period stirs and evenly mixs 3 times, then 10min is centrifuged by 3000r/min, layering occurs, and upper strata is ovum
Yellow antibody extract solution B, separates standby；
3) two kinds of mixing of extracting mode yolk liquid
Yolk antibody extract solution A obtained by step 2 is mixed into obtain mixed liquor with B by equal weight proportions, as monitoring is anti-with yolk
Body sample；
4) hemagglutination test
On Microhemagglutination plate, from the 1st row row of Kong Zhi 12 hole, 25 μ l PBS are added per hole, draw the antigen of certain multiple dilution
25 μ l be added on the 1st row hole, removed after being sufficiently mixed 25 μ l to the 2nd row hole, the like do equivalent doubling dilution to the 11st row hole,
11st row hole discards 25 μ L；The 12nd row hole is set up for red blood cell is compareed；Per the μ l PBS of Kong Zaijia 25；0.75% chicken is respectively added per hole
The μ l of red cell suspension 25；Put immediately and mix 1min on microoscillator, or hand-held blood-coagulation-board mixes 1min around circle；At room temperature
Effect 30min, or act on 60min at 4 DEG C；Observe for several times, until the red blood cell of control is all precipitated as a round dot, by blood
Solidifying plate is inclined, and observation red blood cell whether there is teardrop sample sagging, according to blood clotting spectral discrimination result；Hemagglutinative titer is higher than 211When can continue
Increase the hole count of dilution, with the hemagglutinative titer that the antigen greatest dilution for complete aggegation occur is the antigen, now hemagglutinative titer
It is 2n, n>11, the HAU of antigen is calculated further according to initial dilution；And according to the hemagglutinative titer of the antigen for determining, calculate 4
The extension rate of individual HAU antigen, prepares 4 HAU antigens；
5) hemagglutination-inhibition test confirms with potency
On Microhemagglutination plate, from the 1st row row of Kong Zhi 12 hole, 25 μ l PBS are added per hole；
By the mixed liquor of step 3, take the μ l of volume 25 in the 1st arrange hole, after mixing from left to right order doubling dilution to the 11st row hole,
Finally discard 25 μ l；The 1st row row of Kong Zhi 12 hole adds 25 μ l containing 4 antigens of HAU respectively；Microoscillator is put immediately
Upper mixing 1min, or hand-held blood-coagulation-board mixes 1min around circle；30min is acted at room temperature, or acts on 60min at 4 DEG C；Often
Hole adds the μ l of 0.75% chicken erythrocyte suspension 25；The rearmounted effect 40min at room temperature of mixing acts on 60min at 4 DEG C, now compares
Red blood cell be precipitated as a round dot；It is higher than 2 that blood clotting suppresses potency11When can continue increase dilution hole count, with completely inhibit
The maximum dilution multiple of RCA is that the blood clotting of the mixed liquor suppresses potency, determines antibody titer and is plus revisory coefficient
The blood clotting of Yolk antibody alternative serum antibody suppresses potency；
6) main agents and preparation
PBS：
Prepare 20 times of PB：Weigh 5.157g Na2HPO4.12H2O and 0.874g NaH2PO4.2H2O, plus distilled water is to 100ml；Match somebody with somebody
1 times of PBS of system：Measure 20 times of PB of 50ml, add 8.5g NaCl, plus distilled water is to 1000ml；PH value is adjusted with NaOH or HCl extremely
7.2；121 DEG C of high pressure steam sterilization 15min；
0.75% chicken erythrocyte suspension：
The blood of at least 3 SPF cocks of collection or the healthy cock without epidemic disease antibody is mixed with isometric red cell preservation Alsever's Solution
Close, wash 3 times with foregoing PBS liquid, with foregoing PBS be made into 0.75% after washing red with 1000r/min centrifugation 10min every time
Cell suspension, 4 DEG C save backup, and described 0.75% is volume ratio.
2. preparation method as claimed in claim 1, it is characterised in that：
Wherein described antigen is H5 subtype avian influenza antigens, in step 3 by equal weight proportions, be Yolk antibody extract solution A：
50%, Yolk antibody extract solution B：50%.
3. preparation method as claimed in claim 1, the Alsever's Solution formula in abovementioned steps 6 is：
Red cell preservation Alsever's Solution：Claim glucose 2.05g, sodium citrate 0.8g, citric acid 0.055g, sodium chloride 0.42g, plus steam
Distilled water adjusts pH value to 100ml, after dissolving to 6.1,115 DEG C of high pressure steam sterilization 20min, and 4 DEG C save backup.
4. preparation method as claimed in claim 1, it is characterised in that：
The blood clotting that the step 5 is obtained suppresses potency, adds revisory coefficient, and revisory coefficient receives vaccine, immunization period, immune programme for children
Deng influence, revised potency suppresses potency as the H5 subtype avian influenzas blood clotting of Yolk antibody alternative serum antibody.
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| CN107328944A (en) * | 2017-06-14 | 2017-11-07 | 王琴 | A kind of blood clotting and hemagglutination-inhibition test screening technique |
| CN113504367A (en) * | 2021-06-02 | 2021-10-15 | 威海市环翠区动物疫病预防控制中心(威海市环翠区动物卫生检疫中心) | Hemagglutination inhibition test detection method for avian influenza and newcastle disease |
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| CN113504367A (en) * | 2021-06-02 | 2021-10-15 | 威海市环翠区动物疫病预防控制中心(威海市环翠区动物卫生检疫中心) | Hemagglutination inhibition test detection method for avian influenza and newcastle disease |
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